The bed nucleus of the stria terminalis (BNST) plays an important role in fear and stress, and has been implicated in anxiety disorders including posttraumatic stress disorder (PTSD). The BNST contains a collection of sub-nuclei delineated by cytoarchitecture, molecular and neurochemical features. Dorsal and ventral subregions in anterior BNST contain substantial populations of neurons that express the stress-associated neuropeptide, corticotropin-releasing hormone (CRH) known to modulate BNST-related anxiety-like behaviors. Most of what we know about intrinsic and extrinsic BNST circuit connections comes from classic anatomical studies. These studies tend to have low spatial and cell type resolution. Further, there is little work on the functional synaptic connections within the BNST. Thus many aspects of the circuit organization are not well understood. We propose to use recent technological advancements in genetic cell targeting, molecular and viral tracing, and functional circuit mapping to determine the synaptic circuit organization of specific BNST neuron types with a focus on CRH+ neurons. We hypothesize that CRH-expressing neurons differentially govern neural signal input/output transformations in anxiolytic and anxiogenic promoting subregions of the BNST, and that distinct circuit connectivity differences between these cell types can be mapped to determine their specific roles in regulating stress behavior responses. To test our hypothesis, we will first map global and local circuit connections to CRH+ neuronal types in putative anxiolytic and anxiogenic subregions (Aim 1). New genetically modified rabies-based tracing will be used to map monosynaptic global circuit connections in the intact brain to these selected BNST neurons. To functionally verify the results of the anatomical rabies tracing studies, physiological input characterization of local and long-range connections will be determined by laser scanning photostimulation and channelrhodopsin (ChR2)-assisted circuit mapping. Following input mapping, we will map anatomical and functional output projections of CRH+ neuronal types using anterograde directed viral tracing and ChR2-assisted circuit mapping (Aim 2). This will establish CRH+ projection targets in hypothalamic regions related to the regulation of hypothalamus-pituitary-adrenal (HPA) axis activity. In addition, behavioral analysis of the function of CRH+ neuron types will test how these cell types in specific BNST subregions are integrated in larger functional networks and how they contribute to control of stress responses (Aim 3). This will be achieved by specific optogenetic activation in parallel with physiological and behavioral measures. Together, these studies will generate new maps of CRH+ neuronal circuit wiring in anxiogenic and anxiolytic subregions of the BNST. This research will advance our understanding of BNST-centered neural mechanisms to better understand and treat pathological anxiety disorders.